B/B-like fragment targeting for the purposes of photodynamic therapy and medical imaging

ABSTRACT

The invention provides compositions and methods for use in delivering a substance of interest to a targeted cell. The substance of interest is associated with a targeting fragment of a toxin molecule or a lectin that specifically binds to a cell surface receptor, such as the B subunit of an A/B type toxin molecule. The substance of interest may be a photosensitizing agent, in which case the cell (e.g. a cancer cell) may be killed by exposure to light after delivery of the agent. Alternatively, the substance of interest may be a visualizing agent that enhances visualization of the targeted cell.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention generally relates to the use of a targetingfragment of a toxin or lectin molecule for the delivery of a substanceof interest to cells. In particular, the invention provides acomposition comprising a targeting fragment of a toxin molecule and asubstance of interest, and methods for use of the composition. Moreparticularly, the substance of interest may be a photosensitizing agentfor use in targeted cell killing, or a visualizing agent for use inidentifying cell surface receptors of interest.

[0003] 2. Description of Related Art

[0004] The “holy grail” of research in the battle against cancer hasbeen the development of a magic bullet to selectively kill cancerouscells while leaving normal cells untouched. Standard, therapeuticapproaches to the treatment of cancer include surgery to remove thecancerous tissue (if the tumor is well defined and localized),radiotherapy, chemotherapy or combinations of these methods. Frequently,when a cancerous tumor is removed, a significant portion of surroundingtissue is also removed to ensure that the majority of cancerous cellsare eliminated. In some cases, an entire organ is removed, even thoughportions of the organ are still healthy. In spite of such radicalprocedures, cancer cells may spill into body cavities and remain behindto proliferate. Further, portions of a tumor may be difficult to discernor difficult to access. The ability to accurately target cancerous cellsfor destruction while leaving normal, healthy tissue intact would be amajor step forward in the treatment of this disease.

[0005] One form of therapy that is currently gaining acceptance for thetreatment of hyperproliferating tissues is photodynamic therapy (PDT).Based on the discovery made over years ago that rapidly growing cellstreated with certain chemicals will die when exposed to light, PDT iscurrently being used to treat several different types of cancers andnon-malignant lesions. There appears to be a selective affinity andretention of photosensitizers in hyperproliferating tissue. Commonly, apatient is injected with a photosensitizer (PS) molecule that spreadsthroughout the body. There is then a waiting period during which the PSmolecules accumulate in the target tissue, and are eliminated from mostnon-target tissue. Light is then used to illuminate a mass of tumorcells and activate the PS molecules to produce singlet oxygen, therebykilling cells and tissue in the area. The use of PDT to treat esophagealcancer, lung cancer and macular degeneration is currently beingevaluated in clinical trials. One of the theoretical advantages of PDTis that tissues unexposed to light will not be affected. However, inreality, the rate of clearance of the photosensitizer from normal tissueis highly variable. Thus, while success rates of treatment with PDT areso far impressive, deleterious side effects such as skin sensitivity tolight for four to six weeks have been observed. In addition,inflammation of the treatment site resulting in shortness of breath andcoughing has been observed as a result of PDT treatment of lung andesophageal cancers.

[0006] Attempts have been made to optimize PDT treatments. U.S. Pat. No.6,058,937 to Dorion et al., the disclosure of which is incorporatedherein by reference, presents a method for shortening the waiting periodafter administration of PDT to a tissue. The technique is limited,however, to highly vascularized tissue. Rather than destroying thetissue itself, PDT is used to destroy the vasculature that nourishes thetissue and thus indirectly causes tissue death. The majority ofmolecules do not readily penetrate cell membranes. Methods forintroducing molecules of interest into the cytosol of living cells aredisclosed in U.S. patent application Ser. No. 5,876,989 to Berg et al.,the disclosure of which is incorporated herein by reference. Themolecules to be released into the cell's cytosol are first taken up inendosomes, lysosomes or other cell compartments together with aphotosensitizing compound. Light activation of the photosensitizingcompound is then used to rupture the membranes of the cell compartments.The contents of the ruptured compartments (including the molecule ofinterest) are released into the cell cytosol without killing themajority of the cells. This invention thus utilizes PDT as a mechanismfor releasing a drug (such as gelonin, a ribosome inactivating protein)from endosomes/lysomes to the cell compartment where the drug iseffective (cytosol). The method does not provide a method of cellkilling by PDT.

[0007] Kraus et al. in U.S. Pat. No. 6,160,024 describe chemical linkersto connect an energy emitting compound to a photosensitizing molecule,thereby providing an internal chemically-activated light source foractivation of the photosensitizer. The method was employed to destroyvirus-infected or tumor cells. However, this and other known PDT methodsgenerally lack specificity. Their use results in whole bodysensitization to illumination and the attendant side effects.

[0008] A need, therefore, exists for a way to enhance the specificity ofPDT in order to avoid whole-body light sensitivity.

[0009] In order to enhance the specificity of cancer therapies,researchers have attempted to take advantage of the many biochemical andphysiological changes that occur during cancer cell transformation. Someof these changes include the presence of cell-surface molecules whencells become cancerous. This has led to the use of antibodies coupledwith toxic compounds to selectively bind to the surface of cancerouscells thereby killing those cells. However, this approach also haslimitations that include the heterogeneous uptake of the toxin by thetumor cells, the slow elimination of the antibody-toxin complex from theblood system, and the cross-reactivity of the antibody with normaltissue.

[0010] The differential expression of many cell-surface molecules inhuman cancerous cells has been well studied and thoroughly documented.One such molecule is globotriaosylceramide (also known as Gb₃, CD77 andp^(k) antigen). The Gb₃ glycosphingolipid is normally expressed inseveral tissues including intestinal epithelium, kidney epithelium, andendothelial cells, in addition to being found in human milk as aglycolipid. Gb₃ is also expressed in a fraction of germinal center Blymphocytes, and traces of Gb₃ are found in red blood cell membranes ofmost individuals. Gb₃ is strongly expressed in the red blood cellmembranes of p^(k) blood type individuals (0.01% of the population). Theover-expression of this cell-surface receptor has been documented inovarian cancer, Burkitt's lymphoma (non-Hodgkin's lymphoma), breastcancer, brain cancer, gastric cancer, and testicular cancer. It wouldnot be unreasonable to predict that the over-expression of Gb₃ may occurin many other types of cancer, as well.

[0011] The Gb₃ receptor (intestinal) is targeted by the bacterial toxinproteins belonging to the verotoxin family of bacteriotoxins thatincludes the Shiga toxins and Shiga-like toxins. Bacterial (Shigelladysenteriae and Escherichia coli) production of these toxins leads todisorders such as food poisoning, dysentery, hemorrhagic colitis, andhemolytic uremic syndrome. It is not the actual bacterial infection, butthe production of the toxin molecules that leads to the diseasesymptoms. The bacteriotoxins, both Shiga and Shiga-like, are comprisedof two protein components, a catalytic A subunit and a pentameric Bsubunit. The catalytic A subunit is a potent N-glycosidase that inhibitsprotein synthesis once inside a cell. The B subunit array is responsiblefor targeting specific cells expressing Gb₃ on their surface byrecognizing and binding the Gb₃ receptor. Several issued patents takeadvantage of the targeted specificity of verotoxins to localizedpositions on cancerous cells.

[0012] Verotoxin 1, or the pentameric B subunit of verotoxin 1,administered in a non-lethal amount has been effective in treatingmammalian neoplasia, including ovarian, brain, breast, and skin cancersas described, for example, in Lingwood et al., U.S. Pat. No. 5,968,894,the disclosure of which is incorporated herein by reference.

[0013] Gariepy in U.S. Pat. No. 5,801,145, the disclosure of which isincorporated herein by reference, describes the treatment ofnon-Hodgkin's lymphomas by a related ex vivo method. Taking advantage ofthe fact that Shiga toxin or Shiga-like toxin-1 selectively binds toCD77+ cells, according to this method bone marrow cells are treated withthe toxins to specifically kill CD77+ cells prior to a bone marrowtransplant.

[0014] While some of these methods utilize cell targeting via specificcell surface receptors, they lack the benefits of PDT.

[0015] An indispensable component of cancer treatment protocols is theability to detect and locate, i.e. to visualize tumors. The noninvasivevisualization of cancerous tumors is technically challenging and has ledto the development and application of numerous modalities. These includeX-ray, CT scan, nuclear scan, PET scan, MRI, ultrasound and numerousmodifications that are used to visualize tumors. These techniques areused routinely for general biomedical imaging with each having distinctadvantages and disadvantages. Overall, the most significant disadvantageof these techniques is the lack of sensitivity to detect smallabnormalities. As such, a high level of skilled expertise is required bypersonnel to identify a small cancerous tumor. The detection andidentification of a cancerous growth is paramount to achieving apositive outcome for the patient. The ability to readily distinguishbetween cancerous cells and normal cells would be of tremendous benefitto an oncologist or clinician.

[0016] Thus, there is also an existing need for a method that allowsspecific detection, localization, and visualization of cancerous cells.A methodology that provides noninvasive imaging of tumors and simplevisual discrimination of cancerous versus normal cells would be highlybeneficial.

[0017] Therefore, the principle need is for a vehicle and method fordelivery of substances of interest specifically to targeted cells thatprovides all of the above referenced advantages without thedisadvantages associated with each taken individually.

SUMMARY OF THE INVENTION

[0018] It is a principle objective of the present invention to provide amethodology of delivering molecules of interest to cells. The methodinvolves associating a substance of interest with a targeting fragmentof a toxin or lectin molecule. The targeting fragment mediates thecellular delivery and internalization of the substance of interest. Thesubstance of interest will be internalized by cells possessing the cellsurface receptor for which the targeting fragment is specific.

[0019] In one aspect, the present invention targets a cell surfacereceptor (CD77, Gb₃) identified as being over expressed in ovariancancer cells, Burkitt's lymphoma cells, breast cancer cells, gastriccancer cells, and testicular cancer cells. The receptor is naturallyexpressed in intestinal epithelium, kidney epithelium and endothelialcells, in addition to being found in human milk. This receptor isnaturally targeted by the B subunit of the bacterial protein SLT. Oncebound to the receptor via the B subunit, the entire protein isinternalized by the cell. The B subunit thus functions to “deliver” thecatalytic A subunit of SLT to the cell. By coupling a substance ofinterest to the B subunit of SLT (SLT-B) in lieu of the A subunit, it islikewise possible to deliver the substance of interest to cellspossessing the Gb₃ receptor.

[0020] In one embodiment of the present invention, the substance ofinterest is a photosensitizing agent. For example, the photosensitizingagent chlorin e6 (Ce6) has been well studied and used as a model systemfor photodynamic therapy. The instant invention describes coupling Ce6with SLT-B to affect cell killing using light.

[0021] In one embodiment of the instant invention, the substance ofinterest that is attached to the targeting fragment and delivered to atarget cell is a visualizing agent. The resulting targetingfragment/visualizing agent composition would be useful for identifyingcell surface receptors of interest in patients and clinical samples.This application includes the identification of cell surface receptorsin both pathogenic (e.g. cancerous) tissue and normal tissue. Examplesof suitable clinical samples include but are not limited to biopsysamples, blood samples, bone marrow samples, and the like.

[0022] For the practice of this aspect of the invention, a compositioncomprising a targeting fragment of a toxin or lectin molecule and avisualizing agent are provided to a patient, or to a clinical sample,and the cell surface receptor of interest is then located in the patientor the clinical sample by imaging the visualizing agent after thetargeting fragment has bound to the receptor of interest. Those of skillin the art will recognize that many types of imaging exist that could beutilized in the practice of this aspect of the invention, including butnot limited to such procedures as scintigraphy (nuclear scanning), x-rayor CT scans, magnetic resonance imaging (MRI), and the like. Anysuitable type of imaging may be utilized in the practice of the presentinvention so long as the visualizing agent that is delivered via thetargeting fragment can be detected.

[0023] In one embodiment of the present invention, the cell surfacereceptors that are identified by the practice of the present inventionare associated with cancer cells and identification of the cell surfacereceptors allows visualization of the cancer cells. However, those ofskill in the art will recognize that the visualization of cellsdisplaying such receptors would be advantageous in many circumstances.Examples include but are not limited to the visualization of tissues(normal or diseased) prior to or during surgical procedures,localization of specific cell types, monitoring cell location, and thelike. The method of the present invention may be utilized to identifyany cell surface receptor of interest, so long as the receptor bindswith specificity to a targeting fragment of a toxin or lectin molecule.

[0024] The practice of this aspect of the present invention wouldprovide a distinct advantage in, for example, identifying and locatingtumors or other cancerous cells and thus for planning treatmentprotocols. The method could aid in the assessment of metastasis, or beuseful during surgery to remove tumors. For example, the practice of themethod of the present invention would render cancerous tissues visibleand distinguishable from normal tissue. It would thus be possible to bemore conservative with respect to removal of tissue that is notcancerous, thereby minimizing the loss of healthy tissue by the patient.

[0025] It is yet another objective of the present invention to provide amethodology for the direct visualization of cancerous cells bycomplexing a fluorescent molecule to the B fragment, illuminating withan appropriate light and observing the light emitted. This methodologywould be used, for example, during biopsy or surgical procedures.

[0026] Another objective of the present invention to provide apredictable method of detecting normal expression patterns in intestinaland other tissues. Because the Gb₃ receptor is expressed in tissue otherthan tumor cells, there would be areas of localization of a conjugateregardless of the presence of tumor cells. However, the localization inintestinal tissue or elsewhere would be predictable based upon knownnormal expression patterns. This embodiment of the invention could beused for imaging of normal intestinal, kidney and endothelial tissues(vasculature) for example to detect changes in expression that mayresult from physiological changes associated with conditions such asdisease, pregnancy, administration of drugs, aging, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1A and B. Affinity chromatography. Galabiose-agarose affinitychromatography of Vibrio Cholerae 0395-N1/pSBC54 periplasmic extract(panel A), and Cy3-SLTB used as standard (panel B).

[0028]FIG. 2. SDS-PAGE gel of SLTB. Electrophoresis was run on 15%polyacrylamide gels at a constant current of 20 mAmps. Lanes 1 and 4represent molecular weight standards; lane 2 represents the unboundpolymyxin B extract; lane 3 represents the Shiga-like toxin B fragment(SLTB) after affinity chromatography purification.

[0029]FIG. 3. Binding and uptake of Ce6-SLTB conjugate in Vero cells.Fluorescence images of Vero cells incubated with mixed (covalent andabsorbed) Ce6-SLTB conjugate at 0, 1, 2, and 4 hours of chase.

[0030]FIG. 4. Binding and uptake of Cy3-SLTB in Vero cells. Fluorescenceimages of Vero cells incubated with Cy3-SLTB at 0, 1, 4, and 18 hours ofchase.

[0031]FIG. 5A and B. Ce6-SLTB Localizes to Mitochondria and SecretoryOrganelles: Colocalization of Mito Tracker® Green FM and Ce6-SLTB inVero cells. Panel A:

[0032] MitoTracker® Green FM fluorescence; Panel B: Ce6 fluorescence.

[0033]FIG. 6A and B. Ce6-conjugate concentration dependent cell death.Vero cells grown on 35 mm glass bottom gridded dishes were incubated for18 hours with varying concentrations of the indicated preparation,followed by irradiation as described in Material and Methods. 4 hoursafter irradiation, the extent of cell death was determined as described.6A: ▪=Ce6; =Ce6-SLTB mixed conjugate; ▴=Ce6-SLTB absorbed; X=Ce6 darkcontrol; ⋄=Ce6-SLTB mixed conjugate dark control; ∇=Ce6-SLTB absorbeddark control;

=no Ce6 dark control; ◯=no Ce6 illuminated control. 6B: □=Ce6-SLTB mixedconjugate; Δ=Ce6-SLTB absorbed.

[0034]FIG. 7. Cell Killing is Restricted to Area Illuminated.Fluorescent images of dead and live Ver cells exposed to Ce6-SLTB (mixedpreparation) and then irradiated. Panels A and B represent images takenat 0 hours after irradiation, and Panels C and D represent images taken0.5 hours after irradiation. Panels A and C correspond to calceinfluorescence in live cells, and panels B and D correspond to ethidiumhomodimer-1 fluorescence in dead cells.

[0035]FIG. 8. Kinetics of cell killing: Quantitation of cell death byCe6-SLTB (mixed preparation) photosensitization. Y axis is % dead cells;X axis is time in hours.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0036] Applicant's have discovered methods for selectively delivering asubstance of interest to targeted cells. The method involves providingto cells a composition comprising two moieties: 1) a targeting fragmentof a toxin or lectin molecule and 2) the substance of interest. By “atargeting fragment of a toxin or lectin molecule” we mean the portion ofa toxin or lectin molecule that binds with specificity to a receptorlocated on the surface of a cell, i.e. the targeting fragment is aligand for the cell surface receptor. Such a targeting portion of amolecule may also be termed a “fragment” or “subunit” of the molecule.Those of skill in the art will recognize that in some cases, the portionof a toxin or lectin molecule suitable for use in the present inventionwill be a subunit of a multimeric (oligomeric) molecule, the subunitbeing encoded by a gene that is distinct from that of the othercomponents of the holotoxin or lectin. The holotoxin or lectin isassembled post-translationally, and the targeting subunit may beobtained in a variety of manners, including isolation of the holotoxinor lectin followed by separation of subunit components, cloning of theDNA encoding the targeting subunit and production via recombinant DNAtechnology, synthetic production of the subunit by peptide synthesis,and the like. In other cases, the targeting portion of a toxin or lectinmolecule may be a “fragment” of the entire toxin or lectin molecule,i.e. the part of the polypeptide chain that represents the targetingportion is contiguous with the rest of the molecule and forms part ofthe toxin or lectin polypeptide chain. In this case, the entire toxinmolecule is translated from a single mRNA (as the result of beingencoded by a single gene, or as the result of mRNA splicing). The“targeting fragment” may be obtained as a distinct entity for use in thepresent invention by such methods as, for example, proteolysis of thetoxin molecule, cloning of the DNA that encodes the targeting portion ofthe toxin polypeptide, synthetic production of the targeting fragment bypeptide synthesis, and the like. In addition, the “targeting fragment”itself may be comprised of a single polypeptide chain, or of multiplepolypeptide chains associated with each other e.g. by covalent,hydrophobic, or ionic interactions, and the like, i.e. the targetingfragment may, in and of itself, be oligomeric.

[0037] By “binds with specificity” we mean that the Kd will be in therange of approximately 10⁻¹⁰ to 10⁻²⁰ M⁻¹ , and more preferably in therange of approximately 10⁻¹² to 10⁻¹⁷ M⁻¹. By “substance of interest” wemean a substance that is associated with the targeting fragment and thatpossesses a desired activity. For example, the substance of interest maybe a photosensitizing agent or a visualizing agent. The association ofthe substance of interest with the targeting fragment allows delivery ofthe substance of interest to the targeted cell. Without being bound bytheory, it is believed that the associated substance/targeting fragmentconjugate binds to the cell surface receptor of a targeted cell via thetargeting fragment. Following binding, the substance of interest is“piggy-backed” into the cell via the targeting fragment of the conjugateby receptor mediated endocytosis. However, for some purposes, theconjugate may function equally well by binding to the cell surfacereceptor without internalization. Further, the substance of interest mayenter the cell by means other than receptor mediated endocytosis, e.g.by passive diffusion.

[0038] In some embodiments of the instant invention, the targetingfragment originates from a toxin molecule. In other embodiments, thetargeting fragment originates from a lectin molecule. The name lectincomes from the Latin word legere, which means “to select”. The term wascreated by W. C. Boyd to designate plant agglutinins that had bloodgroup specificity (Kilpatrick, 2000). The biochemical basis of thelectin agglutination reaction with erythrocytes and other cells is therecognition and binding of the lectin to terminal and internalcarbohydrate structures in cell surface glycoconjugates. Lectins arewidespread in nature and are not limited to plants, they are found inanimals, bacteria and viruses. A definition that is broadly accepted inthe field describes lectins as carbohydrate-binding proteins that arenot involved in carbohydrate metabolism and do not belong to any of themain immunoglobulin classes (Kilpatrick, 2000).

[0039] In some embodiments of the instant invention, the substance ofinterest may be, for example, a photosensitizing agent. In this case,the conjugate compositions of the instant invention may be used to carryout selective cell killing by delivering the photosensitizing agent to acell where it is then internalized by the cell. Subsequent exposure ofthe cell to light activates the photosensitizing agent and causes celldamage or death. The cells to which the composition of the presentinvention are provided and which are subsequently exposed to light maybe either in vivo or ex vivo. In other embodiments of the instantinvention, the substance of interest may be, for example, a visualizingagent. The visualizing agent is delivered to a cell via the conjugate.The conjugate is bound to the cell via the targeting fragment moiety,thus providing a method to identify cell surface receptors of interest.(Only those cells possessing the receptor of interest will bind theconjugate). Use of the method permits visualization by various imagingtechniques of cells which posses cell surface receptors that bind thetargeting fragment moiety of the conjugate. Cells to which thevisualizing agent are delivered and which are subsequently imaged may beeither in vivo or ex vivo.

[0040] Those of skill in the art will recognize that many types oftoxins and lectins exist, the targeting fragments of which may beemployed in the composition and methods of the present invention.Potentially useful toxins and lectins include but are not limited tothose presented in Table 1. TABLE 1 Protein Toxins that Possess CellSurface Receptor Targeting Fragments Toxin Name Origin Cell SurfaceReceptor Potential Target Abrin A/B plant non-reducing terminalsarcomas, leukemias toxin galactose-containing glycoconjugates A/B heatEscherichia gangliosides GM1, brain, nerve, labile coli GD1b, GM2intestinal tissue toxins Botulinum Clostridium gangliosides GT1b, brainand nerve toxin botulinum GQ1b tissue (bacterial) Cholera Vibrioganglioside GM1 brain and nerve toxin cholerae tissue (bacterial) HelixPlant lectin terminal breast cancer pomatia alpha-GalNAc Jacalin orPlant lectin TF antigen, Gal-beta1- gastric, pancreatic, Jackfruit3/4Glc/GalNAc- and mammary alpha terminal epitope cancer; ofglycoconjugates malignant oral lesions Peanut Plant lectin terminalGal-beta1- colonic agglutinin 3/4Glc/GalNAc-alpha adenocarcinoma, (PNA)(TF antigen) ulderative colitis, meningiomas Ricin Ricinus non-reducingterminal colon cancer cells toxin communis galactose-containing (plant)glyconjugates A/B plant toxin Sambucus A/B plant non-reducing terminalcolon cancer nigra toxin alpha-2-6 sialic acid (SNA-1) residues inglycoconjugates Tetanus Clostridium gangliosides GT1b, brain and nervetoxin tetani GD1b, GQ1b; sialic tissue; melanomas (bacterial) acidcontaining glycoconjugates Ulex Plant lectin Fuc-alpha1-2-Gal-vasoformative europeaus beta1-3/4GlcNAc tumors (e.g. (UEA-I) (bloodgroup H/O) angiosarcomas) and blood vessel invasion on thyroid tumorsViscumin A/B plant non-reducing terminal ovarian cancer toxingalactose-containing glycoconjugates

[0041] Those of skill in the art will recognize that the nomenclatureused to designate the targeting fragment portion of a toxin moleculewill differ from toxin to toxin. In one embodiment of the presentinvention, the targeting fragment component of the composition of thepresent invention is the B subunit of an A/B type toxin molecule. By“A/B type toxin molecule” we mean an oligomeric or multimeric proteinwhere subunit B is responsible for binding at the cell surface of targetcells and thus delivering the toxic A subunit to the interior of thecell.

[0042] In one embodiment of the present invention, the A/B type toxinmolecule is a verotoxin molecule. The verotoxins are a family ofmultimeric bacteriotoxins that includes the Shiga toxin (ST) andShiga-like toxins (SLTs). Shiga toxins are produced by the bacteriumShigella dysenteriae type 1, and the Shiga-like toxins are produced byvarious strains of Escherchia coli. Shiga-like toxins include types I(SLT-I) and II (SLT-II). The primary structure of SLT-I is very similarto ST, differing by a single amino acid substitution in the A subunit.The B subunits are identical. SLT-I and SLT-II share only about 56%amino acid sequence homology. SLTs specifically target intestinal cells,and production of these toxins by E. coli typically cause symptomsassociated with food poisoning.

[0043] The A and B subunits of toxin molecules of this type each havedistinct functions. The catalytic A subunit is a potent glycanase thatcleaves the N-glycosidic bond at A-4324 in 28S ribosomal RNA, and thuscausing inhibition of protein synthesis. The B subunit functions todeliver the A subunit to the targeted intestinal cells. The B subunit ofverotoxins binds specifically to target cells by recognizing and bindingto the glycosphingolipid cell surface receptor globotriaosylceramide(Gal-alpha-1-4Gal-beta-1-4Glc-Cer, or “Gb₃”).

[0044] Subsequent to binding of the B subunit of the toxin to Gb₃, theentire A/B toxin molecule is internalized by the cell by endocytosis andtransported to the endoplasmic reticulum. The A subunit is thentranslocated to the cytosol where it exerts its effect.

[0045] The A subunit of a verotoxin molecule is made up of a single Achain monomer. The B subunit is a pentamer made up of five B monomers.The B subunit monomers are capable of spontaneously assembling into anactive pentamer in the absence of the A subunit. The resulting B subunitpentamer is capable of binding to the Gb₃ receptor and is taken up bythe cell in the same manner that the intact A/B toxin would be.

[0046] Applicant has discovered that the targeting fragment of a toxinmolecule is amenable to chemical modification and that the resultingchemically modified targeting fragment binds to cells possessing anappropriate cell surface receptor. In particular, applicants havediscovered that the B subunit of Shiga-like toxin type 1 is amenable tochemical modification by attachment of a substance of interest and,without being bound by theory, it appears that the resulting chemicallymodified B subunit binds to and delivers the substance of interest tocells possessing the Gb₃ receptor.

[0047] By “chemical modification” of the targeting fragment we mean theassociation of a substance of interest to the targeting fragment to forma conjugate. The association may be covalent (e.g. the attachment of aporphyrin-type photosensitizer to an amino acid residue such ascysteine, glutamic acid or aspartic acid, or by a carbodiimide reactionto lysine residues), or non-covalent (e.g. via hydrophobicinteractions), or ionic via salt bridges between charged groups on theside chains of the amino acid residues of the targeting fragment and thesubstance of interest. Further, the targeting fragment may be chemicallymodified in other ways including but not limited to the association of avisualizing agent via iodination, radiolabeling with a radioactiveisotope (e.g. I¹²⁵, Tc⁹⁹, and the like), or the chelation ofparamagnetic substances (e.g. gadolinium or iron oxides). Modificationsmay also include the association of a substance of interest byabsorption. Further, the association may be of more than one form, e.g.the conjugate may be comprised of a targeting fragment with a substanceof interest associated both covalently and via absorption. In addition,a single molecule or a plurality of molecules of a substance of interestmay be associated with the targeting fragment. If a plurality ofmolecules of a substance of interest are associated with the targetingfragment, those molecules may be the same or different, i.e. a singletargeting fragment may have associated with it both a photosensitizingagent and a visualizing agent. Those of skill in the art will recognizethat many such modifications exist and can be utilized in the practiceof the present invention. All such modifications are intended to bewithin the scope of present invention, so long as the resulting modifiedtargeting fragment is capable of binding to an appropriate cell surfacereceptor. The instant invention provides a composition comprising atargeting fragment of a toxin or lectin molecule and a substance ofinterest. In the composition, the association of the substance ofinterest to the targeting fragment may be of any type (covalent, ionic,absorptive, and the like). Further, the composition may be “mixed” inthat the conjugates present in the composition may differ in the type ofassociation between the targeting fragment and substance of interest.For example, the composition may be comprised of a mixture ofconjugates, in some of which the association between the targetingfragment and the substance of interest is covalent, and in others ofwhich the association is via absorption.

[0048] In other embodiments, the modification may be the “attachment”via genetic engineering of another polypeptide of interest to thetargeting fragment. This type of attachment may create a chimericprotein molecule comprised of the targeting fragment moiety and at leastone other polypeptide of interest. The targeting fragment and thepolypeptide of interest may be translated from a single mRNA. Those ofskill in the art are well acquainted with techniques for geneticallyengineering such chimeric proteins. Alternatively, another polypeptideof interest may be “attached” to the targeting fragmentpost-translationally via covalent or non-covalent (e.g. hydrophobic orionic) means. Examples of polypeptides that could be utilized to modifythe targeting fragment moiety of the conjugate include but are notlimited to green fluorescent protein (GFP), including various iterationsof the protein (such as those in which λ maximum of absorbance oremission has been altered, or in which various control elements orrestriction sites have been introduced or removed from the coding DNA,resulting in alterations in the translated mRNA) for enhancedvisualization of the targeted cells. Other examples include redfluorescent protein (Ds-Red from Clontech, Palo Alto Calif.), Vitality™hrGFP (from Stratagene, La Jolla, Calif.), luciferase, and the like.Those of skill in the art will recognize that many polypeptides existwhich could be attached to the targeting fragment in order to form achimeric conjugate for use in the practice of the present invention. Allsuch chimeric conjugates are intended to be encompassed by the presentinvention, so long as the chimera is capable of binding to anappropriate cell surface receptor.

[0049] In one embodiment of the present invention, the targetingfragment is a B subunit from Shiga-like toxin (SLTB) type 1. However,those of skill in the art will recognize that many A/B type toxinmolecules exist, the B subunits of which would be suitable for use inthe practice of the present invention. Examples include but are notlimited to: B fragment of Escherichia coli heat-labile enterotoxin, Bfragment of abrin, B fragment of viscumin, and B fragment of Sambucusnigra. Further, those of skill in the art will recognize that manymodifications of a B subunit can be made for any of a variety ofreasons, and that all such modified forms of the subunit may be utilizedin the practice of the present invention. For example, amino acidsubstitutions (conservative or non-conservative), additions or deletionsmay be made in order to, e.g. optimize the binding affinity of thesubunit for its receptor, to alter the stability or solubility of thesubunit, to facilitate the construction of a chimeric conjugate, todecrease the size of the molecule in order to decrease antigenicity, orto add targeting sequences, and the like. If the B subunit is clonedinto a vector, changes may be made to the nucleic acid sequence encodingthe subunit in order to, for example, introduce convenient restrictionsites, or to foreshorten or lengthen the coding sequence for any reason.Any such modification of a B subunit is intended to be encompassed bythe term “B subunit”, so long as the B subunit so modified stillfunctions as described in the practice of the present invention.

[0050] In one embodiment of the present invention, the composition ofthe present invention is comprised of a targeting fragment associatedwith a photosensitizing agent. A “photosensitizing agent” (or“photosensitizer”) is a substance that, upon exposure to light, ispromoted to an excited state, and transfers its energy to a receptormolecule in the environment. The photosensitizer drops to ground statewhile exciting the receptor molecule.

[0051] In the case of photodynamic therapy, the photosensitizertransfers energy to oxygen molecules. Oxygen in its ground state is atriplet (T), but when excited by photosensitization, is promoted to asinglet state (S). Singlet oxygen is very reactive, oxidizing membranecomponents in a manner that causes damage or death to living cells. Theattachment of the photosensitizing agent to the targeting fragment maybe carried out, for example, by absorption (i.e. through non-covalentbonds, for example, via hydrophobic interactions) or by covalent orionic binding, or by a combination of one or more modes of association.An example of covalent binding would be the covalent attachment of thephotosensitizer to the targeting fragment by a carbodiimide reaction.

[0052] In one embodiment of the present invention, the photosensitizerhas a porphyrin structure. Porphyrins are cyclic conjugatedtetrapyrroles such as chlorophylls and hemoglobin. In a preferredembodiment of the present invention, the porphyrin-type photosensitizeris chlorin e6 (Ce6). However, those of skill in the art will recognizethat many types of photosensitizers exist that would be suitable for usein the practice of the present invention. Examples include but are notlimited to: metal phtalocyanines, hypocrellins, hypericin, purpurins,furanocoumarins, chalcogenopyrylium dyes, quinolones, and the like (seeTable 2).

[0053] The selection of a photosensitizing agent is based on severalcriteria. For example, if the targeted cells are to be illuminated invivo, an appropriate photosensitizing agent would be one that has anabsorbance wavelength maximum of at least about 600 nm in order to allowfor deep penetration of the targeted tissue, e.g. a tumor mass. However,if the illumination is carried out ex vivo (as might be the case forexample, in purging targeted cells from a cell sample that was to bereintroduced into the body) photosynthesizing agents with shorterabsorbance maxima might be preferable. In addition, those of skill inthe art will recognize that photosensitizing agents may possess multipleabsorbance maxima and may thus be useful both in vivo and ex vivo.Examples of appropriate photosensitizing agents are given in Table 2.TABLE 2 Photosensitizers and Corresponding Absorbance WavelengthsAbsorbance Wavelength Photosensitizer Type Maxima (nm) 5-Aminolaevulinicacid (ALA) protoporphyrin IX 400, 650 precursor Benzoporphyrinderivative porphyrin 692 monoacid ring A (BPD-MA) Chalcogenopyryliumdyes chalcogenopyrylium 592-675 (thio-, seleno-, telluro- dyes pyrylium)Furanocoumarines (psoralen, furanocoumarines 320-360 xanthotoxin,angelicin) Hypocrellins A/B perylenquinone 658 Hypericin anthraquinone658 Lutetium (III) texaphyrin porphyrin 732 Malachite green isosulphanblue 628 derivative Mono-L-aspartyl chlorin e6 porphyrin 664 Photophrinporphyrin 400, 650 Phthalocyanine tetrasulfonate porphyrin 672 (Zn(II)or Al(II)) Quinolones (spafloxacin, quinolones 330-360 lomefloxacin,enoxacin, ofloxacin, ciprofloxacin) Sn (IV) etiopurpurin dichlorideporphyrin 659 Temporfin (meso-tetra(m- porphyrin 652 hydroxyphenylchlorin)

[0054] The cell killing methods of the present invention are selective.The selectivity occurs on two levels. First, the ligand moiety of theconjugate binds only to specific cell surface receptors. The conjugatewill not bind to cells that do not contain such specific receptors. Thisaspect of the invention takes advantage of the observation that manytypes of cancer cells over-express certain cell surface receptors, e.g.ovarian cancer cells, Burkitt's lymphoma cells, breast cancer cells,brain cancer cells, gastric cancer cells, and testicular cancer cellsover-express the Gb₃ receptor. While it is true that some normal cells(e.g. intestinal tissue cells) also possess Gb₃ receptors and willtherefore to a limited extent accumulate conjugate, the over-expressionof Gb₃ in cancer cells will ensure a bias in the accumulation of thephotosensitizer in cancer cells compared to normal cells. Further, thesecond level of specificity (described below) will attenuate thepotential for damage to normal cells.

[0055] The second level of specificity is that activation of thephotosensitizer (and subsequent cell damage) will occur only uponexposure to light. When the targeted cells are in vivo (i.e. locatedinternally within the organism), they will be exposed to light only whenlight of an appropriate wavelength is deliberately introduced into theenvironment, for example, during a studied surgical procedure using, forexample, optical fibers. For endoscopic use, optical fibers would bethreaded through a catheter or endoscope, allowing for small incisionswhile delivering a focused beam of light. When the targeted cells are exvivo, it would be possible to shield the cells until light of thewavelength that would activate the photosensitizing agent could bepurposefully administered. Many companies (such as Coherent MedicalGroup, Coherent Inc., Palo Alto, Calif.), manufacture productsspecifically designed for the production of narrow wavelengths of lightrequired for medical use. Those of skill in the art are acquainted withand will recognize that many such products exist. For example, gaslasers as well as LEDs are commercially available and capable ofproducing the requisite light. Any appropriate means of illuminating thetarget cells that results in activation of the photosensitizer moleculewithin the target cells, so that injury or death of the target cellsresults, may be utilized in the practice of the present invention. Forexample, of such methods of illumination, see Bellnier, D. et al. 1999.

[0056] The composition of the present invention may be administered forthe purpose of selective cell killing by any of several suitable meansthat are well-known to those of skill in the art. For example,intramuscularly, intravenously, intratumorally, orally, and the like.Due to the intrinsic specificity of the targeting fragment of thecomposition, administration may be systemic. As discussed, while somecell types other than those targeted for killing may also internalizethe conjugate, since they will not be exposed to light, they will not bedamaged or killed. The composition may be administered in any of avariety of suitable forms, including forms that include additionalcomponents such as buffers, stabilizers, and the like, which areappropriate to the means of administration. The exact form, dosage andfrequency of administration will vary from case to case and will dependon factors such as the nature and stage of the disease being treated(e.g. size and location of a tumor), characteristics of the patient(e.g. overall health, age, weight, gender and the like), and otherfactors such as ancillary treatments (chemotherapy, radiotherapy, andthe like). The details of administration are best determined by askilled practitioner such as a physician. Further, the details ofadministration are normally worked out during clinical trials. However,the approximate dosage range will be from about 0. 1 to 10 mg/kg, andmore preferably from about 0.25 to 1.0 mg/kg.

[0057] Likewise, the dose or frequency of illumination of the targetcells will vary from case to case, but will generally be in the range of25 -200 J/cm2 light dose, 25-200 mW/cm2 fluence rate (see Ochsner, 1997,the contents of which is incorporated herein by reference in entirety).

[0058] In an ideal situation, the practice of the method of the presentinvention will result in the death of the targeted cells, e.g. cancercells. However, those of skill in the art will recognize that themethods of the instant invention would also be useful even if the cancercells were not killed outright. Other potential benefits could includeattenuation of the cancer cells that would make them more susceptible toother types of cell killing such as chemotherapy or radiotherapy. (Andindeed the methods of the instant invention may be practiced inconjunction with other therapeutic measures.) Similarly, abrogating ordestroying the ability of the cancer cells to proliferate would also beof benefit, whether or not the cancer cells were killed outright.

[0059] The present invention encompasses methods of use of thecomposition of the present invention to selectively kill cancer cells.Types of cancer cells which may be selectively killed by the methods ofthe present invention include but are not limited to those thatover-express the Gb₃ receptor, e.g. leukemia cells, ovarian cancercells, Burkitt's lymphoma cells, breast cancer cells, gastric cancercells, testicular cancer cells, and the like. The practice of thepresent invention may be utilized to combat cancer of any type in whichthe cancer cells over-express a specific cell surface receptor, and forwhich an appropriate targeting fragment exists that can be suitablymodified by the attachment of a photosensitizing agent.

[0060] While the method of the present invention may be used for theselective killing of various types of cancer cells, other cellularpopulations may be targeted as well. For example, kidney, intestinal,endothelial cells, or cells infected by a pathological agent such as avirus or bacterium, may also be targeted. Any cell populationcharacterized by the unique or biased expression of a cell surfacereceptor, or which can be isolated so that an appropriate wavelength ofimpinging light can be selectively directed to the targeted cells, andfor which an appropriate targeting fragment exists that can be suitablymodified by the attachment of a photosensitizing agent, may beselectively destroyed by exposure to light by the methods of the presentinvention.

[0061] In another embodiment of the present invention, the substance ofinterest is a visualizing agent. In the furtherance of this and otherobjectives, several different approaches could be used in concert withexisting modalities to aid in the identification of cell surfacereceptors of interest and thus to permit visualization of, for example,tumors.

[0062] It is an objective of the present invention to facilitate nuclearscans. In the furtherance of this and other objectives, the B subunit ofSLT-B is radioactively tagged for use in localizing the isotope to atumor mass. This would allow visualization of a tumor mass using wellknown scintigraphic imaging techniques. It would also assist in thedetection of metastatic cancer (primary tumor cells that have moved toanother region of the body). Examples of currently used radioactiveelements include, but are not limited to, technetium-99, iodine-125 andthallium-201. The attachment of these radioactive tracers to the Bfragment can be accomplished using standard radiology chemistrytechniques. The preparation and guidelines for administration of suchcompounds is best determined by a skilled practitioner such as aphysician or radiologist (American College of Radiologist Standards,Tumor Scintigraphy-1996).

[0063] It is yet another objective of the present invention to provide amethodology suitable in X-rays or Computed Tomography scans. In thefurtherance of this and other objectives, iodine or a comparable atom isused as a contrasting agent. Iodine is currently the most widely usedcontrast agent for X-ray and CT scans. The attachment of iodine to the Bfragment will provide a positive contrast and assist in thevisualization of a tumor using these two modalities Iodination of the Bfragment tyrosines can be easily accomplished using well knownchemistry. (Hunter and Greenwood, 1962; and Greenwood, et al., 1963).The preparation and guidelines for administration of such compounds isbest determined by a skilled practitioner such as a physician orradiologist (Pediatric and Adults Thoracic Computed Tomography, AmericanCollege of Radiology, standards. 1998).

[0064] It is yet another objective of the present invention is toprovide a methodology in Magnetic Resonance imaging. In the furtheranceof this and other objectives, paramagnetic contrast agents such asgadolinium or iron oxides are used to alter the environment of H atomsand create a contrast. Gadolinum is the most widely used contrast agentfor MR imaging. Again, it is possible to attach metal complexes to the Bfragment and achieve an enhancement of imaging for a tumor. The covalentattachment of gadolinium and gadolinium complexes to proteins is wellknown (Niemi et al.,1991; Weiner et al., 1997). The use of iron oxidenanoparticles for general contrasting purposes has been described(Schmitz et al., 1997). The preparation and guidelines foradministration of such compounds is best determined by a skilledpractitioner such as a physician or radiologist (Magnetic ResonanceImaging, American College of Radiology, standards. 1996)

[0065] It is yet another objective of the present invention to provide amethodology for the direct visualization of cancerous cells bycomplexing a fluorescent molecule to the B fragment, illuminating tissuewith appropriate light and observing the light emitted. The practice ofthe method of the present invention would render the cancerous tissuesvisible and distinguishable from normal tissue. This methodology wouldbe used during biopsy or surgical removal of a cancerous tumor. It wouldthus be possible to be more conservative with respect to removal oftissue that is not cancerous, thereby minimizing the loss of healthytissue by the patient. The appropriate light sources and glasses withnecessary filters for illumination and visualization are commerciallyavailable (GFP spectacles, Biological Laboratory Equipment Maintenanceand Services Ltd., Budapest, Hungary). There are numerous fluorescentmolecules that could be utilized in the present invention. Severalcharacteristics of a suitable fluorescent molecule would be theexcitation and emission wavelengths (Stokes radius), molar extinctioncoefficient, quantum yield, and chemical reactivity. Molecular Probes,(Eugene, Oreg.) specializes in the design and manufacture of fluorescentmolecules for a variety of purposes. The covalent attachment offluorophores to proteins is accomplished by commercially availablechemical techniques utilizing various functional groups (amines,carboxylic acids, thiols). Sigma Chemical Co. (St. Louis, Mo.) andMolecular Probes (Eugene, Oreg.) sell such kits. The fluorescentmolecule could also be proteinaceous in nature and attachment to thetargeting fragment may be via the creation of a chimeric protein, asdescribed above.

[0066] The following Examples serve to illustrate various embodiments ofthe instant invention.

[0067] However, they should not be construed so as to limit theinvention in any way.

EXAMPLES Materials and Methods

[0068] Materials.

[0069] Shiga-like toxin I, fragment B (SLTB) was obtained from Vibriocholerae 0395 N1 containing the SLTB-encoding plasmid pSBC54. PolymyxinB nonapeptide was purchased from Sigma-Aldrich, St. Louis, Mo. Galabioseagarose resin was purchased from Calbiochem, La Jolla, Calif. Chlorin e6(Ce6) was obtained from Porphyrin Products, Inc., Logan Utah. FluorLink™ Cy3 reactive dye (bisfunctional NHS ester) was purchased fromAmersham Pharmacia Biotech, Piscataway, N.J. MitoTracker Green FM®,Calcein AM and Ethidium homodimer-1 were purchased from MolecularProbes, Eugene Oreg. (3-(Dimethylamino) propyl)-3-ethyl-carbodiimidehydrochloride (EDC) and 1 -cyclohexyl-3(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate (CMCS) were purchased from Aldrich, Milwaukee,Wis. Sulfo-N-hydroxysuccinimide (sulfo-NHS) and Coomassie Plus ProteinReagent were obtained from Pierce, Rockford, Ill. LB media, LB agar,minimum essential media (MEM), phenol red free-DMEM/F12 (1:1)/15 mMHepes media, fetal calf serum (FCS), penicillin, and streptomycin werepurchased from Gibco BRL, Grand Island, N.Y.

[0070] Bacterial Cultures and Preparation of Periplasmic Extract.

[0071] Vibrio cholerae 0395 N1 (pSBC54) was plated in LB agar containing100 μg/ml ampicillin and 100 μg/ml streptomycin, and grown overnight at37° C. Individual colonies were picked and cultured overnight at 37° C.,in 10 ml of LB media with 100 ,g/ml ampicillin and 100 μg/mlstreptomycin (Acheson et al., 1993). Cultures were then transferred into1 liter of LB media/100 μg/ml ampicillin and 100 μg/ml streptomycin andincubated for 14 h at 37° C. Bacteria was pelleted by centrifuging at5,000×g for 20 min at 4° C. The pellet was suspended in PBS andcentrifuged under the same conditions. Bacterial periplasm was releasedby suspension of the pellet in 5 ml of 2 mg/ml polymyxin Bnonapeptide/PBS, incubation for 25 min at 4° C., followed bycentrifugation at 14,000×g, for 20 min at 4° C. The periplasmic extractwas decanted from the bacterial pellet and stored at -70° C.

[0072] Purification of SLTB by Affinity Chromatography. SLTB waspurified from the periplasmic extract by affinity chromatography ongalabiose-agarose resin (2ml of resin in 1×3 cm column).

[0073] Briefly, the galabiose agarose column was equilibrated inphospahate buffered saline (PBS)/0.02% azide. Two ml of the periplasmicextract were applied to the column and incubated for 15 min at roomtemperature. The column was washed with 10 ml of PBS/azide, and boundSLTB was eluted with 10 ml of 0.1 M glycine HCL, pH 2.5. To minimizedenaturation of SLTB, the 0.1 M glycine fractions were collected intotubes containing neutralizing 1 M Tris. Protein content in fractions wasmonitored by absorbance at 280 nm.

[0074] Bound fractions were pooled, dialyzed in 10 mM sodium phosphatebuffer, pH 7.4 and concentrated down to 1-2 mg/ml of protein using aCentricon Plus-80 centrifugal filter device, MWCO 10,000 (Millipore,Bedford, Mass.). Purity of the bound fractions was assessed byconventional SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteinconcentration was measured by absorbance at 280 nm or Coomassie PlusProtein reagent.

[0075] Ce6-SLTB and Cy3-SLTB Preparations.

[0076] Cy3-SLTB conjugate—Preparation was made following manufacturer'sspecifications with some modifications. Briefly, to one vial of Cy3reactive pack (1 mg), 0.4 ml of 1.8 mg/ml SLTB in 10 mM sodium phosphatebuffer, pH 7.4 were added. The vial was covered with aluminum foil, andincubated overnight at room temperature in a tube rotator device. Freedye was separated from Cy3 -SLTB on a PD10 Sephadex G-25M column(Amersham Pharmacia Biotech, Piscataway, N.J.). The final proteinconcentration of the Cy3-SLTB conjugate was 0.17 mg/ml and the molar dyeto protein ratio was 1:1.

[0077] Mixed (absorbed and covalent) Ce 6-SLTB preparations—Ce6 carboxylgroups were activated with 1 -cyclohexyl-3 (2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCS) and reaction of lysineresidues on SLTB (Aklynina et al, 1997; Faulstich and Fiume, 1985), in aratio of SLTB :Ce6:CMCS of( 1:400:800). Briefly, 1ml of 2 mg/ml SLTB in10 mM sodium phosphate buffer, pH 7.4 was added to a vial containing Ce6and CMCS. The vial with reactants was vortexed gently, wrapped withaluminum foil, and incubated overnight at room temperature in a tuberotator device. The Ce6-SLTB preparation was separated from free Ce6 bygel filtration on a G-75 Sephadex column (1.3×48 cm). Ce6 and proteinwere monitored by absorbance at 400 nm and 280 nm, respectively.Fractions eluting in the void volume of the column were pooled togetherand dialyzed against 8 liters of 10 mM sodium phosphate buffer, pH 7.5.Alternatively, Ce6 (10 mg) was derivatized with (3-(dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride (EDC) andsulfo-N-hydroxysuccinimide (sulfo-NHS) in 0. 1M MES, 0.5 M NaCl, pH 6.0(0.5 ml) (Staros et al., 1986) in the ratio of Ce6:EDC:sulfo-NHS of1:4:2.7, for 30 minutes at room temperature, followed by addition of 1ml of 2 mg/ml SLTB in 10 mM sodium phosphate buffer pH 7.5 and overnightincubation at room temperature in a tube rotator device. Ce6-SLTB wasseparated from free dye as described above. The protein concentrationsof mixed Ce6-SLTB preparations were measured using Coomassie PlusProtein reagent, using as a blank a solution of free Ce6. Theconcentration of Ce6 in Ce-SLTB was determined by absorbance at 400 nmand 280 nm in comparison to standard curves for free Ce6 at bothwavelengths. The concentration of Ce6 in the preparations was correctedfor quenching by SLTB by measuring abosorbance at 400 nm and 280 nm in5% sodium dodecyl sulphate (SDS) solutions of Ce6 and Ce6-SLTB.Quenching of absorbance of Ce6 in Ce6-SLTB was 50% at 400 nm, whereasabsorbance of Ce6 at 280 nm was not affected by protein quenching. Themixed Ce6-SLTB preparations contained both Ce6 covalently linked to SLTB(Ce6-SLTB-covalent) and Ce6 absorbed to SLTB (Ce6-SLTB-absorbed). Themolecular weight of the covalently bound conjugate was approximately 6.2kD as calculated from SDS-PAGE. About 89% of total Ce6 was absorbed and11% was covalently coupled to SLTB.

[0078] Absorbed Ce6-SLTB Preparations—Ce6 (5 mg) was mixed with 0.8mg/ml SLTB in 10 mM sodium phosphate buffer, pH 7.4, followed byincubation overnight, in the dark, in a tube rotor device. Free Ce6 wasseparated from bound Ce6 to SLTB by G-75 Sephadex chromatography asdescribed above. Excluded fractions from the G-75 chromatography werepooled together and dialyzed. Concentration of the Ce6-SLTB-absorbedconjugate was determined as described.

[0079] Cell Culture.

[0080] Wild type Vero cells (ATCC CCL 81) were cultured in minimumessential medium (MEM), containing 10% fetal calf serum (FCS), 100units/ml of penicillin and 100 μg/ml of streptomycin. Cells were kept ina 37° C. incubator, 5% CO₂ /air atmosphere.

[0081] Photodynamic Cell Killing

[0082] Vero cells were grown in 35 mm glass bottom gridded microwelldishes (MatTek Corp., Ashland, Mass.) to 60% confluence. Cells werewashed 3 times with cold MEM media containing 0.1% bovine serum albumin(BSA) and 100 units/ml of penicillin and 100 μg/ml of streptomycin.Cells were then incubated with Ce6, Ce6-SLTB preparations (0.1 -2.0 μM)in MEM/0.1% BSA/100 units/ml penicillin/100 1μ/ml streptomycin at 37°C./5% CO₂/air for 18 h.

[0083] Before irradiation cells were washed 3 times with warm phenol redfree-DMEM/F 12 (1:1)/15 mM Hepes/10% FCS/100 units/ml penicillin/100μg/ml streptomycin and kept in the same media during irradiation. Glassbottom microwell dishes were placed in the microscope stage (AxiovertS100TV, Zeiss, Jena, Germany), with the center of the dish positionedperpendicular to the center of the condenser light (using grids on thecoverslip as a guide), and irradiated with the microscope halogen lamp(100 W, 12 V, 9.8 W/mm²) set at 6 V for 3 minutes. Followingirradiation, cells were washed twice with warm MEM/10% FCS/100 units/mlpenicillin/ 100 μg/ml streptomycin, and incubated in the same media for15 minutes to 18 hours at 37° C./5% CO₂/air, before scoring dead/livecells.

[0084] Cell Viability Assay

[0085] The fluorescent probes calcein AM and ethidium homodimer-1(Molecular Probes, Eugene, Oreg.) were utilized to detect the presenceof live and dead cells, respectively. Calcein AM itself isnon-fluorescent and permeable to membranes; it becomes fluorescent whenhydrolyzed by esterases in live cells. Ethidium homodimer- 1 penetratesthe damaged membranes of dead cells, accumulating in the nucleus, whereits fluorescence is enhanced by DNA binding. Briefly, irradiated disheswere washed 3 times with warm phenol red free-DMEM/F12 (1:1)/15 mMHepes/0.2% BSA/100 units/ml penicillin/100 μg/ml streptomycin, followedby incubation with a solution containing 2 μM calcein AM and 4 μMethidium homodimer- 1 in the same media, for 35-45 minutes at roomtemperature. Fluorescence was visualized using an Axiovert S100TVinverted microscope (Zeiss, Jena, Germany), equipped with Plan-Neofluar2.5×/0.1 NA, 5×/0.075 NA or 10×/0.3 NA objectives (Zeiss, Jena,Germany). The light source was a 50 W mercury arc lamp and excitationand emission wavelengths were selected with the filter sets HQ480/40,Q5051p, HQ 535/50 for calcein (green fluorescence); and HQ545/30,Q5701p, HQ610/75 for ethidium homodimer-1 (red fluorescence) (ChromaTechnologies, Brattleboro, Vt.). Images were captured using a RoperPhotometrics SenSys charged coupled device (CCD) camera (Tucson, Ariz.).The camera was controlled with IPLab software for Macintosh, Version3.5.5 (Scanalytics, Fairfax, Va.). Live and dead cells were scored bymanually counting green fluorescent cells and red fluorescent cells incaptured images. Cells that were both red and green were considereddead. In addition, live and dead cells were scored by morphology changesin phase-contrast images.

[0086] Binding and Uptake Experiments

[0087] Vero cells grown on 35 mm glass bottom gridded dishes were washed3 times with cold phenol red free-DMEM/F12 (1:1)/15 mM Hepes/0,1%BSA/100 units/ml penicillin/100 μg/ml streptomycin, and then incubatedwith 2 μM Ce6-SLTB or 0.04 μM Cy3-SLTB in the same media, at 4° C. for 1hour to allow cell surface binding without protein internalization.Then, plates were washed twice with warm MEM/10% FCS/100 units/mlpenicillin/100 μg/ml streptomycin and incubated at 37° C./5% CO₂/air fordifferent periods of time. After each chase time, cells were washedtwice with PBS and fixed with 3% formaldehyde. Zero time chase plateswere washed once with cold phenol red free-DMEM/F 12 (1:1)/15 mMHepes/10% FCS/100 units/ml penicillin/100 μg/ml streptomycin, then oncewith PBS, and fixed with 3% formaldehyde. Fluorescence in cells wasviewed with an Axiovert S100TV inverted microscope (Zeiss, Jena,Germany) with a Plan Apochromat 63×/1.4 NA oil objective (Zeiss, Jena,Germany). The filter set D405/20X, 425DCX, E600LP (Chroma Technologies,Brattleboro, Vt.) was used for Ce6, and HQ545/30, Q5701p, HQ610/75(Chroma Technologies, Brattleboro, Vt.) for Cy3. Images were captured asabove.

[0088] Fluorescent Staining of Mitochondria

[0089] Vero cells grown on 35 mm glass bottom gridded dishes werestained with MitoTracker® Green FM, a mitochondria-specific fluorescentprobe. Warm MEM/10% FCS/100 units/ml penicillin/100 μg/ml streptomycincontaining 200 nM MitoTracker® Green FM was added to the dishes andincubated for 45 minutes at 37° C./5% CO₂/air. Cells were washed 3 timeswith warm phenol red free-DMEM/F12 (1:1)/15 mM Hepes/10% FCS/100units/ml penicillin/100 μg/ml streptomycin and then observed on themicroscope as described in Binding and Uptake Experiments, using thefilter set HQ480/40, Q5051p, HQ 535/50. Double labeling with Ce6-SLTBand MitoTracker® Green FM were carried out by incubating cells with 2.0μM Ce6-SLTB in MEM/0.1% BSA/100 units/ml penicillin/100 μ/mlstreptomycin at 37° C./5% CO₂/air for 18 h. Cell were then washed threetimes with warm MEM/10% FCS/100 units/ml penicillin/i 100 μg/mlstreptomycin and incubated with 200 nM Mitotracker® Green FM in the samemedia and for 45 min at 37° C./5% CO₂/air. Loading media was removed andthe cells were washed 3 times with warm phenol red free-DMEM/F12(1:1)/15 mM Hepes/10% FCS/100 units/ml penicillin/100 μg/mlstreptomycin. Cells were viewed on the microscope as above.

Example 1

[0090] Production and Purification of SLTB

[0091] Recombinant SLTB was produced from Vibrio cholerae 0395 N1transfected with the plasmid pSBC54 that encodes the gene for Shiga-liketoxin I fragment B (Acheson et al, 1993). The yield of SLTB from oneliter of bacterial cultures was approximately 30 mg. SLTB was purifiedfrom the periplasmic extract in a single step by affinity chromatographyon galabiose-agarose as shown in FIG. 1. SDS-PAGE of the boundgalabiose-agarose fraction showed a single band of about 5.6 kDmolecular weight (FIG. 2) consistent with the reported molecular weightof SLTB monomer (Acheson et al, 1993). This example demonstrates thatample quantities of purified SLTB can be produced by this method.

Example 2

[0092] Characterization of Mixed Ce6-SLTB Absorbed/Covalent Preparationsby SDS-PAGE.

[0093] Mixed absorbed and covalently conjugated Ce6-SLTB were producedin incubations using carbodiimide activation of the carboxylic groups onCe6 and reaction with lysine residues on SLTB as described in Materialand Methods. Massively aggregated Ce6-SLTB was removed as an insolubleprecipitate that remained on top of a G-75 Sephadex column. The mixedCe6-SLTB preparation was excluded from the G-75 Sephadex column andcollected in the void volume. Association of Ce6 with the mixedpreparation was stable to extensive dialysis.

[0094] SD S-PAGE analysis of the chromatographed, dialyzed preparationsindicated that they contained both absorbed Ce6 and covalentlyconjugated Ce6-SLT. The Ce6 from Ce6-SLTB-absorbed dissociated from SLTBduring electrophoresis and ran at the “dye” front, whereasCe6-SLT-covalent migrated as a species of about 6.2 kDa, a 0.6 kDaincrease in apparent molecular weight over the starting SLTB (data notshown). Based on Ce6 intensities in SDS-PAGE gels, 89% of the Ce6 in themixture was present as absorbed Ce6-SLTB, and 11% of the Ce6 was presentas covalent Ce6-SLTB.

Example 3

[0095] Delivery of Ce6 to Targeted Cells by Ce6-SLTB Conjugates

[0096] Experiments were carried out in which the ability of Ce6-SLTBpreparations to promote the delivery of Ce6 to targeted cells wasassayed. Vero cells were incubated at 4° C. with either free Ce6 ormixed (absorbed and covalent) Ce6-SLTB preparations (equal quantities ofCe6 in both). The results showed that when cells were incubated withmixed Ce6-SLTB preparations, cell-associated Ce6-specific fluorescencewas readily detected (FIG. 3). In contrast, cells incubated with freeCe6 under the same conditions showed no detectable Ce6-specificfluorescence (data not shown), indicating that the presence of SLTB wasessential for the association of Ce6 fluorescence with the targetedcells.

Example 4

[0097] Comparative Binding and Uptake of Cy3-SLTB and Mixed Ce6-SLTBPreparations.

[0098] Cy3 conjugated SLTB is an accepted covalent tracer for thebinding and intracellular internalization of SLTB protein (Johannes etal, 1997; Girod et al., 1999). Binding of Cy3-SLTB at 4° C. to Verocells gave a rim staining pattern about the edge of individual cells,staining typical of SLTB binding to its cell surface receptor (FIG. 4,Oh) Chase of the cell surface bound Cy3-SLTB for 1-18 hour at 37° Cresulted a pattern of fluorescence consistent with retrograde transportof Cy3-SLTB from the plasma membrane to the Golgi apparatus (GA) and tothe endoplasmic reticulum (ER) as reported elsewhere ( Johannes et al.,1997; Girod et al., 1999). Retrograde transport is the process by whichendocytic vesicles containing surface receptor and bound toxin aretransported from early endosomes all the way to the Golgi and then tothe endoplasmic reticulum. FIG. 4 shows photographs of the Vero cellsafter 0, 1, 4, and 18 hours of chase. This pattern of uptake anddistribution is indicative of receptor-mediated endocytosis.

[0099] In contrast, at 4° C. Ce6-SLTB mixed conjugate showed littlesurface binding to Vero cells as evidenced by a lack of edge staining ofthe cells, and instead showed fluorescence staining over the entire cellbody (FIG. 3). Chasing of mixed Ce6-SLTB at 37° C. from 1-18 hoursresulted in a distinct change in the character of the Ce6 staininginside the cells. Fluorescence inside Vero cells increased, and itappeared to be more localized in thick, tubular organelle structuresthat resembled mitochondria. Photographs taken at 0, 1, 2, and 4 hoursof chase are depicted in FIG. 3.

[0100] This pattern of uptake and distribution is not indicative ofreceptor mediated endocytosis, but rather would be consistent withpassive diffusion of Ce6 across the plasma membrane and into theinterior of the cell. This observation is likely the result of bindingof the Ce6-SLTB-absorbed species to the Gb₃ receptor, followed byrelease of the Ce6 into the plasma membrane and entry of the Ce6 intothe cell via passive diffusion across the membrane.

[0101] This example demonstrates that SLTB functions as an efficientmeans to transport a photosensitizing agent into the cytoplasm of cells.

Example 5

[0102] Mixed Ce6-SLTB Preparation Distribution in Vero Cells

[0103] In order to determine if Ce6 was accumulating in mitochondria,double labeling experiments were carried out in which Vero cells weresimultaneously labeled with the green fluorescent mitochondria marker,MitoTracker® Green FM and exposed to mixed Ce6-SLTB. The pattern ofgreen fluorescence resulting from MitoTracker® Green FM labeling (FIG.5A) and the pattern of red fluorescence resulting from the accumulationof Ce6 (FIG. 5B) was determined, and an overlay comparison of the two(red and green) fluorescence patterns was carried out. The comparisonshowed that most of the Ce6 co-localized with MitoTracker® Green FM inmitochondria. This observation is consistent with passive diffusion ofthe Ce-6 across the plasma membrane, suggesting that the source of theCe6 in mitochondria may be from the Ce6-SLTB-absorbed conjugate species.

[0104] However, Ce6 red fluorescence was also observed, albeit to alesser extent, in the GA and ER of the cells. This is most likely due tothe entry of some Ce6 into the cell by receptor mediated endocytosis,consistent with the delivery of Ce6 to the cells by the (approximately11%) Ce6-SLTB conjugate in which the Ce6 is covalently bound.

[0105] Thus, the nature of the association of Ce6 with SLTB (covalent orabsorptive) appears to determine the final destination of Ce6 inside thecell. Ce6 absorbed to SLTB appears to accumulate in mitochondria,whereas Ce6 covalently linked SLTB localizes in the GA and ER of thecells, and at the cell surface.

Example 6

[0106] Comparison of Mixed-Ce6-SLTB-preparation, Absorbed-Ce6-SLTB andFree Ce6 Dependent Photodynamic Cell Killing.

[0107] Vero cells were treated with varying concentrations of free Ce6,mixed-Ce6-SLTB preparation, and absorbed-Ce6-SLTB, followed byirradiation with a halogen lamp as described in MATERIAL AND METHODS.The results of this experiment, presented in FIG. 6A, showed that bothmixed Ce6-SLTB preparations and absorbed-Ce6-SLTB are significantly moreefficient at photodynamic cell killing than free Ce6. Expressed in molarconcentration of Ce6, the LD50 for absorbed Ce6-SLTB was 0.1 nmol/ml,the LD50 for mixed Ce6-SLTB was 0.6 nmol/ml, and the LD50 of free Ce6was 1.2 nmol/ml. Plotting cell death versus SLTB concentration (FIG. 6B)showed that, at the same protein concentration (0.015 μM), both mixedCe6-SLTB-conjugate and absorbed Ce6-SLTB produced the same degree ofcell death.

[0108] These results clearly demonstrate that both mixed Ce6-SLTBpreparations and absorbed-Ce6-SLTB are significantly more efficient atphotodynamic cell killing than free Ce6. Further, these results confirmthat the targeting (B) fragment of Shiga-like toxin type 1 is aneffective vehicle for delivering a substance of interest, such as aphotosensitizer, to cells containing the Gb₃ cell surface receptor.

Example 7

[0109] Localization and Kinetics of Cell Killing with MixedCe6-SLTB-preparation.

[0110] The localization of cell killing by mixed Ce6-SLTB wasinvestigated by calcein and ethidium homodimer-1 fluorescence. Calceinfluorescence occurs only in live cells, whereas ethidium homodimer-1fluorescence occurs only in dead cells. The results are depicted in FIG.7, panels A-D, where panels A and C correspond to calcein fluorescenceat 0 and 0.5 hours after irradiation, respectively, and panels B and Dcorrespond to ethidium homodimer-1 fluorescence at 0 and 0.5 hours afterirradiation, respectively. As can be seen in Panel A, cells exposed tothe Ce6-SLTB preparation display readily detectable calcein fluorescenceimmediately after irradiation (0 hours), i.e. they are alive. However,as can be seen in Panel C, 0.5 hours after irradiation few living cellsremain within the central, circular area of the field that wasirradiated, whereas the remaining non-irradiated area is still populatedby living cells. Likewise, ethidium homodimer-1 fluorescence of Ce6-SLTBexposed cells shows essentially no dead cells immediately afterirradiation (0 hours, Panel B). However, 0.5 hours after irradiation(Panel D), the circular area of the field that was irradiated (and onlythe circular irradiated area) contains many dead cells. Thisdemonstrates that the killing of cells exposed to Ce6-SLTB is confinedto only those cells which are exposed to light. Cells which are notexposed to light are not affected.

[0111] In order to determine the rate of cell killing after mixedCe6-SLTB-preparation treatment and irradiation of Vero cells, cell death(as indicated by the percentage of dead cells) was assessed at timesranging from 0-18 hours post-irradiation. The results showed thatextensive cell death was evident as soon as 0.25 hours afterirradiation. Quantitation of the data (FIG. 6) revealed greater than 70%cell death only 0.25 hours after irradiation, and close to 95% celldeath two hours after irradiation. After 5 hours post-irradiation, celldeath was essentially 100% and dead cells were observed to detach fromthe dish.

[0112] This example demonstrates that treatment of cells with a mixedCe6-SLTB-preparation followed by irradiation is a rapid and effectivemethod of cell killing.

[0113] While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

References

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[0115] Aklynina, T. V., Jans, D. A., Rosenkranz, A. A., Statsyuk, N. V.,Balashova, I. Y., Toth, G., Pavo, I., Rubin, A. B., and Sobolev, A. S.1997. Nuclear Targeting of Chlorin e6 Enhances its PhotosensitizingActivity. Journal of biological Chemistry. 272: 20328-20331.

[0116] Bellnier, D. et al. 1999. Design and construction of alight-delivery system for photodynamic therapy. Med. Phys. 26: 1552.

[0117] Faulstich, Hl and Fiume, L. 1985. Protein Conjugates of FungalToxins. Methods in Enzymology, 112: 225-237.

[0118] Girod A., Storrie, B., Simpson, J. C., Johannes, L. Goud, B.,Roberts, L. M., Lord, J. M., Nilsson, T., and Pepperkok, R. Evidence fora COP-I-independent transport route from the Golgi complex to theendoplasmic reticulum. Nature Cell Biology, 1: 423-430. 1999.

[0119] Greenwood, F. C., Hunter, W. M. and Glover, J. S., 1963.Biochemical Journal, 89, 114-23.

[0120] Hunter, W. M. and Greenwood, F. C., 1962. Nature, 194, 495-6.

[0121] Johannes, L., Tenza, D., Antony, C., and Goud, B. 1997.Retrograde transport of KDEL-bearing B fragment of Shiga toxin. Journalof biological Chemistry, 272, 19554-19561.

[0122] Kilpatrick, D. C. 2000. Introduction to Animal Lectins. In:Handbook of Animal Lectins: Properties and Biomedical Applications, pp.1-10. J. Wiley and Sons, LTD, Chichester, England.

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We claim:
 1. A method for identifying a cell surface receptor ofinterest in patients and clinical samples, comprising a) providing tosaid patients or clinical samples a composition comprising a targetingfragment of a toxin or lectin molecule and a visualizing agent; andlocating said cell surface receptor in said patient or clinical sampleby imaging said visualizing agent after said targeting fragment of saidtoxin has bound to said cell surface receptor.
 2. The method of claim 1wherein said targeting fragment of a toxin or lectin molecule is a Bfragment of an A/B type toxin molecule.
 3. The method of claim 2 whereinsaid B fragment of an A/B type toxin molecule is selected from the groupconsisting of B fragment of Shiga-like toxin type-1, B fragment ofEscherichia coli heat-labile enterotoxin, B fragment of abrin, Bfragment of viscumin, and B fragment of Sambucus nigra.
 4. The method ofclaim 1 wherein said visualizing agent is an X-ray/CT contrast agent. 5.The method of claim 4 wherein said X-ray/CT contrast agent is iodine. 6.The method of claim 1 wherein said visualizing agent is an MRI contrastagent.
 7. The method of claim 6 wherein said MRI contrast agent isselected from the group consisting of a paramagnetic atom and aparamagnetic compound.
 8. The method of claim 7 wherein saidparamagnetic atom is gadolinum.
 9. The method of claim 7 wherein saidparamagnetic compound is iron oxide.
 10. The method of claim 1 whereinsaid visualizing agent is selected from the group consisting of afluorescent molecule and a fluorescent protein.
 11. The method of claim1 wherein said visualizing agent is a radioactive atom.
 12. The methodof claim 1 wherein said cell surface receptor is selected from the groupconsisting of Gb₃, GM1, GM2, Gd1b, GT1b, TF antigen, non-reducingterminal galactose, N-acetylgalactosamine, alpha 2-6 sialic acid, andalpha 1-2 fucose containing glycoconjugates.